Continued alcohol use is facilitated by alcohol's positive (rewarding) properties, whereas its negative (aversive) properties serve to limit consumption. Dependence is associated with greater tolerance to ethanol's aversive properties than its rewarding properties, which is thought to promote continued consumption. The rostromedial tegmental nucleus (RMTg) exerts inhibitory control over mesolimbic dopamine neurons and is critically involved in signaling the aversive properties of drugs of abuse. The prelimbic (PrL) subregion of the prefrontal cortex shares several critical functional similarities with the RMTg including facilitating aversion learning and responding to aversive stimuli. Despite these similarities, the projection from the PrL to the RMTg has been largely ignored and remains an open area of investigation. This K99/R00 proposal comprises a comprehensive training and research plan based upon the candidate's (Dr. Elizabeth Burnett) preliminary data identifying a role for the RMTg in signaling the aversive properties of alcohol. During the mentored K99 phase of the award, Dr. Burnett will receive training in cutting-edge laboratory techniques including slice electrophysiology, optogenetics, analysis of synaptic morphology, and virally-mediated trans-synaptic tracing. These techniques will augment her existing expertise in cellular and molecular approaches and behavioral models of alcohol abuse and dependence. Under the guidance of the candidate's mentoring team, which includes the primary mentor (Dr. Judson Chandler) and co-mentor (Dr. John Woodward), the candidate will expand her current studies by testing a novel hypothesis that alcohol-induced plasticity in the PrL-RMTg pathway plays a role in the functional consequences of chronic ethanol exposure. Aim 1 will investigate the effect of selective activation of the PrL-RMTg pathway during dependence-induced escalated and aversion-resistant ethanol intake using in vivo optogenetics in combination with operant behavioral testing. Aim 2 will use whole-cell patch-clamp slice electrophysiology and in vitro optogenetics to examine the effect of chronic ethanol exposure on synaptic plasticity within the PrL-RMTg. Aim 3 will define the PrL-RMTg's expanded neurocircuitry by identifying neurons that synapse onto PrL neurons projecting to the RMTg using virally-mediated trans- synaptic retrograde tracing. Experiments under this aim will also determine the involvement of these newly identified second-order RMTg inputs in chronic ethanol exposure by measuring cFos induction in these neurons during withdrawal. The results of these studies will provide new insights into the role of the PrL-RMTg pathway in promoting escalated alcohol consumption. Improving our understanding of the neurocircuitry involved in mediating the aversive component(s) of alcohol consumption may help uncover new therapeutic targets for the treatment of alcohol use disorders. The training Dr. Burnett will receive during the mentored K99 phase of the award will facilitate her career development and the results obtained during the R00 phase of the award will form the foundation of Dr. Burnett's independent research program.